EXPRESSION CLONING OF A CDNA-ENCODING UDP-GLCNAC-GAL-BETA-1-3-GALNAC-R (GLCNAC TO GALNAC) BETA-1-6GLCNAC TRANSFERASE BY GENE-TRANSFER INTO CHO CELLS EXPRESSING POLYOMA LARGE TUMOR-ANTIGEN
EXPRESSION CLONING OF A CDNA-ENCODING UDP-GLCNAC-GAL-BETA-1-3-GALNAC-R (GLCNAC TO GALNAC) BETA-1-6GLCNAC TRANSFERASE BY GENE-TRANSFER INTO CHO CELLS EXPRESSING POLYOMA LARGE TUMOR-ANTIGEN
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DOI：
10.1073/pnas.89.19.9326
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发表时间：
1992-10-01
影响因子：
11.1
通讯作者：
FUKUDA, M
中科院分区：
文献类型：
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作者：
BIERHUIZEN, MFA;FUKUDA, M
使用中国仓鼠卵巢（CHO）细胞，通过表达克隆方法分离了编码UDP-GlcNAc：Galbeta 1 -3GalNAc-R（GlcNAc至GaINAc）beta1- 6 GlcNAc转移酶（EC 2.4.1.102）的cDNA，该转移酶在O-聚糖中形成关键分支。已在多种生物过程中观察到这种酶的活性增加和O-聚糖核心2结构[Galbeta 1 -3（GlcNAc beta1 -6）GalNAc]的伴随出现，例如T细胞活化和由于Wiskott-Aldrich综合征和AIDS引起的免疫缺陷。由于CHO细胞不表达该酶，因此建立CHO细胞系以稳定表达多瘤大肿瘤（T）抗原，这使得能够进行瞬时表达克隆。因为发现所用抗体最有效地检测附着于白唾液酸的寡糖产物，所以CHO细胞也用白唾液酸cDNA稳定转染。通过使用这种特定的CHO细胞系，从HL-60 cDNA文库中分离编码决定核心2结构形成的蛋白质的cDNA。的cDNA序列预测的蛋白质与II型膜拓扑结构，已被发现的所有其他哺乳动物糖基转移酶克隆日期。推定的催化结构域作为与蛋白A的IgG结合结构域的融合蛋白的表达使我们能够明确地证明cDNA编码核心2 β-1，6-N-乙酰葡糖胺转移酶，该酶负责形成Galbeta 1 -3（GlcNAc beta1 -6）GalNAc结构。未检测到该酶对其他β 1 - 6 GlcNAc转移酶受体的活性。
A cDNA encoding UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GaINAc) beta1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Galbeta1-3(GlcNAcbeta1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Galbeta1-3(GlcNAcbeta1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta1-6GlcNAc transferases.